Influenza virus continues to cause significant illness in humans. Development of new vaccines takes time, and many of the circulating strains are resistant to available drugs. New strategies for identifying anti-viral targets are needed. The virl glycoprotein hemagglutinin (HA) catalyzes membrane fusion, which is crucial for viral entry, and the clustering of HA is necessary for fusion to occur. We recently discovered an association between HA and several host cell actin binding proteins (ABPs) and actin itself in the absence of other viral components. However, the mechanism for this interaction is unknown. Interactions could occur directly through the cytoplasmic domain (CTD) of HA, but the CTD is relatively short, and some literature has found that its removal is inconsequential to viral infection. However, other literature shows a marked effect of removing cysteines in the CTD which serve as palmitylation sites, including failed virus growth, reduction in infectivity, and reduction in te membrane fusion activity of HA. Furthermore, the level of conservation of these cysteines in the CTD is remarkable, suggesting great importance for the segment. The proposed project will attempt to determine the mechanism by which HA interacts with host cell actin. Because certain ABPs are known to bind palmitate groups, we will test the hypothesis that HA palmitylation modulates HA clustering through its interactions with ABPs. HA clustering has also been shown to modulate cell signaling pathways related to actin, and we observe that expression of HA in cells causes actin remodeling. Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a lipid known to modulate cell actin through a number of signaling pathways, and can also bind many of the same ABPs we find to colocalize with HA. The second aim of the project will therefore investigate whether PIP2 plays a role in HA clustering, and specifically in its associations with ABPs and actin. We will use the super-resolution microscopy method fluorescence photoactivation localization microscopy (FPALM) (cited within the scientific description of this year's Nobel Prize in Chemistry) to examine the nanoscale interactions between HA and the actin cytoskeleton under perturbations of HA lipid modifications, cellular PIP2 levels, and cell signaling. Results obtained will help determine the mechanism for influenza interactions with host cell actin, improve understanding of membrane cell biology, and identify new host cell targets for anti-viral therapies.